Apparatus for isoelectric focusing

ABSTRACT

A method for reducing the time required to obtain separation with a great resolution of components in a sample solution by isoelectric focusing consists of applying an electrical field between a pair of electrodes positioned at the top and bottom of density gradient columns in successive steps, a succeeding step being performed with a density gradient column having a smaller cross-section perpendicular to the electrical field than that of the column used in the preceding step.

United States Patent [1 1 Rilbe et al.

APPARATUS FOR ISOELECTRIC FOCUSING Inventors: Svante Harry Rilbe; JarlSune Pettersson, both of Goteborg, Sweden LKB-Produkter AB, Bromma,Sweden Filed: May 2, 1973 Appl. No.: 356,307

Assignee:

US. Cl. 204/299; 204/180 R Int. Cl. B0lK 5/00 Field of Search 204/180 R,180 G, 299',

References Cited UNITED STATES PATENTS 12/1950 Dinsley 204/299 1 Oct.28, 1975 3,453,200 7/1969 Allington 204/299 X 3,498,905 3/1970 Strickler204/299 3,616,455 10/1971 Von Miinchhausen 204/299 3,791,950 2/1974Allington 204/299 X Primary ExaminerJohn l-l. Mack Assistant Examiner-A.C. Prescott [57] ABSTRACT 16 Claims, 5 Drawing Figures US. Patent Oct.28, 1975 Sheet 1 of3 3,915,839

m b a US. Patent Oct. 28, 1975 Sheet 2 of 3 3,915,839

FIG. 3

FIG.2

US. Patent Oct. 28, 1975 Sheet 3 of 3 3,915,839

FIG

APPARATUS FOR ISOELECTRIC FOCUSING The present invention relates to amethod in separation of components of a sample solution by isoelectricfocusing in a focusing chamber which is subject to a direct currentelectrical field, and to an apparatus for accomplishing the method.

isoelectric focusing is an electrophoretic method of separation, basedon differences in isoelectric points of the sample components to beseparated, and which is performed in an electrical current path having avarying pH. Isoelectric focusing is generally characterized in thatafter equilibrium has been achieved, the separated substances arelocated in that point in the pH- gradient which corresponds to theisoelectric point of each substance, respectively, and that eachsubstance there has zero mobility. A survey of isoelectric focusing asseparation method has been given by Haglund in Methods of BiochemicalAnalysis, 19, page 1-104, John Wiley & Sons (1971).

The pH-gradient, requisite in isoelectric focusing, can be obtained intwo principally different ways. Thus, the pH-gradient could beartificial or natural. The artificial pH-gradient is achieved by amechanically obtained mixture in gradually changed proportion ofsolutions of varying pH. The natural pH-gradient is obtained by theinfluence of the electrical field on a number of ampholytical substanceshaving different isoelectric points. Thus, the natural pH-gradient hasthe advantage after having been formed by influence of the electricalfield to be stabilized thereby. The theory of natural pH-gradients wasdiscussed by Svensson in Acta Chem. Scand. 15, page 325-341 (1961), andin Acta Chem. Scand. 16, page 456-466 (1962).

In order to achieve practically a natural pH-gradient, suitable socalled carrier ampholytes are required, the isoelectric points of whichare narrowly and evenly distributed over the desired pH-interval andwhich possess in their respective isoelectric points buffering as wellas electrically conducting properties. The lack of good carrierampholyte was the limitation of this method until such substances weremade available by an invention, made by Vesterberg, disclosed in theU.S. Pat. No. 3,485,736 (corresponding to Swedish Pat. No. 314,227). Thedisclosed mixture of carrier ampholytes are marketed by LKB-ProdukterAB, Bromma, under the Trade Mark Ampholine.

The present invention can be performed by utilization of an artificialor a natural pH-gradient. It is preferred by the performance of thepresent invention to use a natural pH-gradient.

Separation of substances by isoelectric focusing in a naturalpH-gradient is generally described by Svensson in Protides of theBiological Fluids, 15, page 515-522, Elsevier Publishing Company,Amsterdam (1968).

As appears from the mentioned literature some kind of convectionstabilizing measures are required at isoelectric focusing. Whenperforming the present invention the isoelectric focusing is carried outin a density gradient. This method implies that the relatively smalldensity differences depending on temperature differences as well asthose depending on the concentrations of the zone components are drownedin considerable greater, artificial density differences. Generally, thedensity gradient should be parallel or anti-parallel to the appliedelectrical field. Usually, the density of the solution is continuouslyincreasing downwards. This is achieved by using a heavier and a lightersolution, the proportion of the heavier solution increasing downwards inthe column in which the isoelectric preparation is performed, while theproportion of the lighter solution decreases. Both solutions containelectrolytes, the heavier one in addition sugar to an amount of 500grams/liter. The total density difference will amount to about 0.2grams/cm.

Substances which are suitable for separation by isoelectric focusing areampholytes. An important group of such substances are proteins.

A criteria of a good separation is the resolving power of the separationmethod. In isoelectric focusing the term resolving power is directedtowards the smallest difference in isoelectric points of two componentsof the sample solution, which will result in two perceivable zones afterfocusing. ln Acta Chem. Scand., 20, page 820-834 (1966), Vesterberg andSvensson have theoretically deduced and experimentally confirmed thefollowing relationship concerning the resolving power of the isoelectricfocusing method:

where ApI is the smallest difference in isoelectric points, D is thediffusion coefficient of the sample substance, E the electrical fieldstrength, prevailing in the zones, dpH/dx the pH-gradient present in thezones, and du/dpH the slope of the mobility curve (as a function of pH)at the isoelectric point. It should be observed that the pH change perlength unit, not per volume unit, should be put into this relationship.

In order to achieve the best possible resolution, i.e. the smallestpossible ApI, dpH/dx should consequently be given as low a value aspossible. In other words the available natural pH-gradient should bedistributed over as long a distance as practically possible. D anddu/dpH are constant for a given substance, while an increase in Etheoretically should result in an increased resolution. An increase inE, however, will entail an increased heat evolvation in the samplesolution which would imply increased risk of heat convection. Thus, E islimited by the ability of the prevailing density gradient to cover theconvection caused by heat evolvation.

It, at performing an isoelectric focusing, the pH-slope is made veryflat, this will entail that also the density change is distributed overa longer distance, which will decrease its convection stabilizingcapacity. Consequently, the electrical field strength has to be keptsufficiently low in order that the generation of heat convection isavoided. This will result in low ion migration velocities, which willbring about long separation time. The pH-gradient extended over a longdistance moreover will entail long migration paths for the majority ofthe samples components, which will contribute to a prolonged focusingtime. Consequently, the best possible resolution is achieved at the costof slow focusing.

An apparatus for separation of substances by isoelectric focusing isshown in the above mentioned article of Svensson in Protides of theBiological Fluids, 15, page 515-522. This apparatus, which is marketedby LKB- Produkter AB, Bromma, is a development of an apparatus, whichwas described by Vesterberg, Wadstrom, Svensson and Malmgren in Biochim,Biophys. Acta, 133, page 435-445 (1967). The apparatus is designed forisoelectric focusing in natural pH-gradient and in a density gradient.It is characterized by a relatively tall and narrow column with chilledvertical walls. Experiments in order to determine the resolving power ofthis apparatus were described by Vesterberg and Svensson in Acta Chem.Scand., 20, page 820834(l966). According to this publication it ispossible to obtain a resolving power of ApI 0.02, i.e. the apparatusdescribed permits a very high resolution.-On the other side isoelectricfocusing in this apparatus suffers from the above mentioned disadvantagethat only a relatively low field strength can be applied, resulting inlong separation periods. In the experiments, mentioned above, theisoelectric focusing was continued for 48 hours, but a focusing periodof 24-28 hours is stated as being normal. Moreover, the focusing periodin some instances could be still longer, up to 3 days. Thus, thedescribed apparatus requires very long focusing periods but will inreturn give a very good resolution.

By reducing the migration distances of the sample components thefocusing period could be shortened. Furthermore, the density gradient ismade stronger, whereby a higher field strength and considerably greaterelectrical current can be applied, which will result in greatermigration velocities. Such a technique in isoelectric focusing has beendescribed by Kolin in Methods of Biochemical Analysis, 6, page 259-288(1958). It is stated that, by means of this technique, the separationperiod can be reduced to a few minutes. However, the obtainableresolution has not been specified, lesser been documented, and has to beassumed to be far inferior to that which can be achieved by means of theapparatus, described above. Kolin mentions (loc. cit. page 264, secondparagraph) that a flatter pH- gradient should give an increasedresolution. The considerably longer distance, over which the pH-gradientcan be extended in the above mentioned apparatus, will however intail asuperior resolution, while the focusing also embraces a widerpH-interval.

The isolation of fractions after completed focusing constitutes aspecial problem. Sample zones which are not too narrow, in a column witha relatively small cross-section, can easily be isolated formicropreparative purposes by sucking out through a capillary tube, asthe horizontal inflow to this tube could be restricted to somecentimeter or less. The fractions are also often run off through thebottom of the column, which one then is designed in that way that thecrosssection of the column gradually is reduced to the diameter of theoutlet tube. The last mentioned method is applied in the byLKB-Produkter AB marketed apparatus, described above. The densitygradient then is exerting a stabilizing action against convection and infavour of laminar flow, bringing the fractions to flow out in correctorder and with a minimum of remixing. In the apparatus, described byKolin, the isolation of fractions is said to take place by sucking outthrough a capillary tube, but considerable practical difficulties wouldbe at hand with the very thin sample zones which are obtained atfocusing in said apparatus. Focusing according to Kolins method wouldaccordingly find an application only for analytical purposes as far asthe separated zones in some way can be photographed and thenquantitatively be evaluated.

If it is desired, starting from the apparatus described by Kolin, toincrease for preparative purposes the capacity of the method byincreasing the cross-section of the column with retained strong densitygradient and steep pH-course, sucking out through a capillary tube willbe practically impossible to perform, as the horizontal inflow then willbe so long that the mixing with over and under lying liquid layers willbe inevitable. Kolin has suggested, as described in loc.cit. page277-278, that the separated zones are pressed apart by a plunger whichdiminishes the cross-section of the column, whereby the risks ofremixing by sucking out through the capillary tube undoubtedly arereduced. The inserting of the plunger will not, however, increase theresolution as defined in the expression for ApI above, then theunsharpness of the zones will be magnified to the same extent as theirmutual distances. A reduced diffusive remixing after disconnecting ofthe focusing current is however gained, since the diffusive masstransport is proportional to the cross-section and the concentrationgradient, which are both reduced by introduction of the plunger.Remixing by diffusion is particularly rapid for narrow zones as thewidening of zones is proportional to the square root of the timemeasured from an infinitely narrow zone.

If, for preparative purposes, the cross-section is increased in a veryshort column, in which a great field strength is applied, the generatedheat must be diverted, in order that the density differences, generatedby the heat, should not exceed the artificial density gradient. Philpotshowed in Trans. Faraday Soc. 36, page 38-46 (1940), that radial coolingwill give rise to radial density gradients, which will constitute alimiting factor at the electrophoretical separation. According toPhilpot the heat, generated, should be axially diverted, whereby theartificial density gradient on the other hand can be strengthened.

Further, the apparatus, described by Philpot, hasa the same limitationsas to the isolation of fractions as the above mentioned apparatus,described by Kolin.

Kolins statement, that cooling of the focusing columns should not benecessary, would relate to the very small amounts of sample which wereseparated in the apparatus, described by Kolin, and the consequentlysmall total heat generation in the column.

it is a purpose of the present invention to provide a method for theseparation by isoelectric focusing of components in a sample solution,at which the focusing is accomplished in a short period of time and witha high resolution.

It is another purpose of the present invention to provide a method forseparation by isoelectric focusing of components in a sample solution,at which the focusing is accomplished in a short period of time and witha great resolution over a wide pH-interval.

It is another purpose of the present invention to provide an apparatus,by means of which components in a sample solution can be separated byisoelectric focusing in a short period of time and with a greatresolution,

It is another object of the present invention to provide a method forisoelectric separation of proteins for analytical purposes, at which theseparation is accomplished rapidly and with a great resolution.

It is another object of the present invention to provide a method forisoelectric separation of proteins for preparative purposes, at whichthe separation is accomplished fast and with a great resolution.

It is another object of the present invention to provide a method forisoelectric focusing, in which the focusing is performed in severalsteps with a step-by-step increasing resolution in the separation.

It is another object of the present invention to provide an apparatusfor isoelectric focusing, in which the focusing is performed in severalsteps with a step-bystep increasing resolution in the separation.

The characteristics of the invention are obvious from the claims,following the specification.

The purpose of the invention is accomplished by performing a focusingstep with a mean cross-section of the focusing chamber, perpendicular tothe electrical direct current field which is smaller than thecorresponding mean cross-section in the nearest preceding focusing step.

More closely one focusing step is performed with a greater relationbetween column height, i.e. that distance in the column over which theelectrical field is acting, and cube root of the column volume that inthe nearest preceding focusing step.

The simplest form of a column device having varying mean cross-sectionperpendicular to the electrical field consists of one parallelepipedicalcolumn with the linear measures a, b and c, a b c, in which theisoelectric separation according to the invention can be accomplished inthree steps, the first one with c, the second one with b and the thirdone with a as vertical dimension. The intermediate step can be omitted.Such a column requires different electrodes for different spatialorientations. Therefore, the electrodes have to be detachable.

The invention can also be performed by using a column with verticalwalls made by an elastic material, like rubber, which permits acontinuous varying of the mean cross-section perpendicular to theelectrical field with retained column volume.

The invention can finally be performed by using at least two columnshaving different mean cross-section perpendicular to the electricalfield, situated in sequence, one above another, and provided withconnecting tubes between the top of a lower situated column and thebottom of the next higher situated one. The connecting tubes should bemainly vertical in order to allow, after completed prefocusing in onecolumn, a transport free of convection of the column content to the nextcolumn for fine focusing.

In a column having a large cross-section, a plane bottom and a planetop, the outflow through an outlet tube, situated centrally in thebottom or in the top, will not be sufficient as it will imply ahorizontal fluid transport along a path as long as the radius of thecylindrical column. A lag in the outflow from the peripheral parts ofsuch a column can not be avoided, which will result in a certainremixing of separated components. This can be counteracted by means of avery slow outflow, but then the diffusion will be of increasedimportance, and the essential purpose of the invention to speed up theseparation with retained great resolution cannot be completely realized.

The outflow can be improved by making top and bottom conical, with theoutlet tube situated in the apex of the cone. This is, however, no goodsettlement, as it will entail a higher field strength and currentdensity in the peripheral parts of the column than in its central parts.This will lead to a horizontal temperature gradient followed by ahorizontal density gradient contribution which will easily result inthermical convection.

Consequently, only a very slight conicity could be allowed in the topand in the bottom of the column.

A considerably better way is to completely dispense with an axialoutflow of short and wide columns. In contrast, an outlet tube isapplied at approximately half the height ofa vertical column wall and onthe opposite wall a similar tube for inflow of fluid or air tosubstitute the outflowing column content. After complete prefocusing thewhole column is turned 90 in order that the two tubes will get positionsat the very top and at the very bottom of the column tilted on its edge.The density gradient in the column will then, apart from some lag at thewalls, retain its vertical direction whereby the vertical section of thecolumn will get a geometry which is much more favourable for the fluidtransport out of the column. If the column is cylindrical the two tubesare situated on diametrically opposite points on half the heigth of thecylindrical envelope surface. If the column is parallelepipedical thetwo tubes are situated in two opposite corners of a rectangle making ahorizontal section of the column in focusing position.

The tilting of the column 90 before emptying should be carried out bymeans of suitable mechanical means, preferably by motor operation, andthe tilting should not be carried out faster than during a few minutes.Manual turning could hardly be accomplished sufficiently slowly andcarefully.

The fluid transport from a prefocusing column to a fine focusing columnof the same volume as the prefocusing column could be carried outupwards as well as downwards. At transportation upwards a mainlyvertical connection tube between the uppermost point of the prefocusingcolumn and the bottom of the fine focusing column is utilized. Theupward transport is accomplished by pumping into the bottom of theprefocusing column a fluid with at least as great a density as thegreatest density in the sample solution. The column content is therebylifted with a minimum of disturbance into the originally empty finefocusing column. Alternatively, it is possible without use of a fluidpump to allow the lower connection tube of the prefocusing column to dipinto a supply of a fluid with at least as great a density as thegreatest density in the sample solution, whereupon a suitable vacuum isapplied to the fine focusing column. The column content will then besucked up from the prefocusing column into the fine focusing column. Thetransportation velocity is adjusted to a low value, either by adjustingthe vacuum in the upper column or by throttling of one of the connectiontubes.

At transportation downwards the fine focusing column is first filledwith a fluid with at least as great a density as the greatest density inthe sample solution. Then a mainly vertical tube connection, free ofair, is established between the upper end of the fine focusing columnand the bottom of the prefocusing column. By opening of a bottom valveof the fine focusing column the fluid content of the prefocusing columnis caused to flow down spontaneously into the fine focusing column witha minimum of disturbance.

an empty fine focusing column, as this method gives rise tounnecessarily extensive remixing of separated components.

In certain focusing experiments it occurs that only a limitedpI-I-interval is of interest for fine focusing. Then it could beadvantageous to transfer only this fraction ofinterest into a finefocusing column having a volume, adapted to the volume of the fraction.

The electrode system could be designed in many different ways. To placethe electrodes in separate electrode vessels of considerable volumecompared to the column volume was introduced into the classicalelectrophoresis research by Michaelis in Biochem. Z., 16, page 81 (1909)and meant at that time an important progress as possible disturbinginfluence of electrode reaction products in the column thereby wasprevented. Such disturbing influence could be brought about by ionmigration, by diffusion or by convection from the electrodes to thecolumn. The principle of electrodes in separate electrode vessels couldadvantageously be utilized also in the present invention. In that casethe electrode vessels should be in electrolytically conductingconnection to the column device, one to its top and the other to itsbottom. In order to prevent undesired ion migration throughout thecolumn the anolyte must consist of an acid or of a mixture of acids,while the catholyte must consist of a base or of a mixture of bases.

In a preferred embodyment of the present invention the column content isdelimited from anolyte and catholyte by convection breaking membranes,permeable to electrical current. Such membranes could consist of glassfilter discs, other porous discs, cellophane, relatively thick papermembranes, animal membranes, natural or synthetic gel discs, etc. Thesemembranes should be strictly horizontal and should completely cover thecross-section of the column without leakage.

The use of such membranes implies the advantage that the chilling effectof the cooling system could be exerted on anolyte and catholyte eitherby bringing, by means of a circulation pump, the anolyte and thecatholyte to circulate between the column device and a tube coil,immersed into a cooling reservoir, or by correspondingly bringing thecooling medium to circulate through tubes installed in the anolyte andcatholyte chambers outside the membranes. Such a cooling systemgenerates in the column a vertically directed temperature gradient whichentail diversion of current heat in axial direction. This coolingprinciple, which was recommended by ihilpot in. Trans. Faraday Soc. 36,page 38-46 1940) is of course of greatest importance in very shprt andwide columns, and particularly at their bottom, where the densitygradient will receive a strengthening deriving from the temperaturegradient which is directed upwards. At the top of the column acorresponding weakening of the density gradient is caused. These sideeffects can be compensated when preparing the artificial densitygradient by strengthening the density gradient in the upper part of thecolumn at the cost of a certain weakening in the lower part of thecolumn.

If relatively high concentrations of strong acid and strong base arechoosen, the anolyte and the catholyte will obtain conductivities whichare some lO-powers greater than the conductivities generally prevailingin the column. The connection tubes between electrode vessels and columncould therefore be rather slender and do not require particular cooling.

There are great freedom in the choice of electrode type when using anapparatus having separate electrode vessels. Gasing as well asnon-gasing electrodes could be utilized, as can electrodes of noblemetals, carbon, silver, mercury etc.

In the apparatus, showed by Svensson in Arch. Biochem. Biophys. Suppl.1, page 132l38 (1962), noble metal electrodes were used in directcontact with the top and the bottom of the column. This is possibleowing to the principle of the natural pI-I-gradient. As this electrodetype generates the gases hydrogen and oxygen, Svensson suggests for thebottom electrode a gas outlet tube centrally situated in the cylindricalcolumn. This is simply accomplished in columns with a great quotientbetween height and cube root of volume, i.e. columns with relativelymoderate cross-sections. Attempts to apply the same principle to widecolumns will result in a very uneven current density. An electrodesituated in the bottom ofa wide column therefore mainly has to fill thebottom surface of the column, and then the electrode must not be gasing,as rising gas bubbles should bring about convective disturbance in thecolumn.

In order that an electrode should be non-gasing it is required that atthe anode there is a reducing agent sufficiently strong to preventevolvation of oxygen and at the cathode an oxidation agent sufficientlystrong to prevent evolvation of hydrogen gas. One exception from thisrule is constituted by palladium which in pure form or as a silver alloyis able to absorb large quantities of hydrogen. On the other hand apalladium electrode, saturated with hydrogen, could be utilized as anon-gasing anode as hydrogen in that case is present in the metal as areducing agent. This property of palladium is well known since long, butit was not made use of in electrophoretical apparatus until 1960 byNeihof and Schuldiner (Nature 185, page 526 (1960)). In an importantembodyment of the present invention the hydrogen absorbing ability ofpalladium is also utilized.

The reducing agent, which has to be present at a nongasing anode, couldeither be the metal itself or the anolyte acid or some non-electrolytewhich also is part of the anolyte. An example of the metal itself beingreducing agent is a silver electrode in an anolyte of hydrogen halideacid, yet not hydrofluoric acid. The silver halide, formed at the anodeoxidation, is almost completely unsoluble and will consequently not giverise to any migration of silver ions into the column. Another example isa lead anode in an anolyte environment of sulphuric acid. A thirdexample is mercury in hydrogen halide acid environment, but mercurycould only be used as a bottom electrode.

An example of the anolyte acid as reducing agent is hydrogen bromideacid or hydrogen iodide acid in contact with a noble metal or carbonanode. (Philpot, loc.cit., used ammonium bromide in contact with acarbon anode.) The oxidation at the anode generates free liquid bromide,which however may diffuse into the column and cause damage on proteinsthere. A more harmless, reducing acid is ascorbic acid.

The oxidating agent, requisite at a non-gasing cathode, can also bepresent on the cathode itself or in the catholyte. Silver, covered by anadhering layer of silver halide, is a known example, but such a cathode,when working, will evolve halide ions which will migrate into thecolumn. A bottom electrode of mercury, covered by a layer of calomelworks in a corresponding way. Oxidating, soluble bases are not known.Consequently, if the oxidation agent should be present in the catholyteit has to be a non-electrolyte. Only palladium is able to function asnon-gasing cathode without generating foreign anions which will migrateinto the column.

By way of example the following drawings show a number of possibleembodyments of the invention. The invention should not be restrictedthereto.

In the drawings,

FIGS. 1 and 1a are, respectively, longitudinal and transversecross-sectional elevations of a preferred form of one form of separationchamber;

FIG. 2 is a vertical cross-section of a modified form of prefocusingcolumn combined with a final focusing column;

FIG. 3 is a cross-section of a column similar to FIG. 2 but having amodified cooling system; and

FIG. 4 is similar to FIGS. 2 and 3, but modified to place the finalcolumn below the prefocusing column.

FIG. 1 shows a parallelepipedical combination column 14 comprising afocusing chamber 15 having internal measures a, b, and 0 length units, ab c. Outside the two largest surfaces, ab units or surface, there aretwo cooling jackets 1,2, each provided with two pipe sockets 3,4. Eachof the two smallest walls, internally bc units of surface, is providedwith a threaded opening 5,6 in which alternatively a threaded stopper, athreaded tube or a threaded electrode attachment could be inserted.

In this combination column the attachment of detachable electrodesconstitutes the most difficult problem. Therefore, the constructionshown in the figure is restricted to the realization of currenttransport along the dimensions 12 and a, which then should be verticaldimensions as the current direction has to be parallel or antiparallelto the density gradient. In the first case the column stands on a facehaving the internal measures ac units of surface, and in that positionthe prefocusing is accomplished. In the latter case the column issituated on a face having the internal measures be units of surface, andin this position the final focusing is accomplished. The column couldalso be layed on a face having the internal measures ab units ofsurface, i.e. with c as vertical dimension. This position is suitablyutilized at preparation of the density gradient as described by theInventors in Separation Science 3, page 535 (1968). According to thismethod a small number of solutions having rising density are layered oneunder the other in a column, whereupon the column is turned down to ahorizontal position in order to enlarge the diffusion area as well asthe concentration gradient. The mass transport due to free diffusionthereby is increased to such an extent that only a short while isrequired for development of a continuous density variation in thecolumn.

The problem of the bottom electrode could be solved very easily by usingmercury, which always is spreading over the bottom independent of thespatial orientation of the column. Mercury 7 as an anode is non-gasingif it is present in an environment of hydrogen halide acid or sulphuricacid, as difficultly soluble mercury I salts are formed. Mercury ascathode is non-gasing in an environment of alkali hydroxides due to theformation of alkali metal amalgam. The external connection of a mercuryelectrode is also most simple. A platinum wire 8 will suffice, goingthrough the column wall and arriving at an arbitrary corner of thecolumn. In the figure the lead-through is intended to be bent at anangle and leading through a column face of internal measures ab units ofsurface to make possible that the column is placed upon a column face ofinternal measures ac units of surface.

Every non-gasing electrode must have a large area in order to keep thecurrent density as low as possible. When the column is in prefocusingposition (b vertical) a non-gasing top electrode consequently must havean area of the size of ac units of surface. The most suitable materialis palladium-silver-alloy as cathode and the same alloy, saturated withhydrogen, as anode. Any fluid space between such an electrode and thecolumn wall is non-desirable. Then it remains nothing but to coat thiscolumn wall with palladium-silver-alloy, and then the whole wall has tobe detachable. A constructively simple way to make this is shown in thefigure. The column wall 9 itself, made of plastic, having a T- shapedcross-section, is fastened by means of a number of screws 10 and istightened by means of a O-ring 11. A thin sheet ofpalladium-silver-alloy with the dimensions c X (a+d) units of surface,where d is slightly greater than the thickness of one of the end wallswith internal measure b units of surface, constitutes the electrode 12.The end wall has a cut out slit 13 having the electrode 12 protrudingout of it to make possible to be connected to the voltage source. Aftercompleted prefocusing, the current is disconnected and all the screws 10are loosened. The electrode 12 then can be pulled out whereupon all thescrews 10 are once again tightened. The column then is carefully turnedupright in order that a will become vertical dimension. Through thetreaded opening 6 a platinum wire is introduced, fastened in a threadedelectrode attachment provided with a gas outlet hole, whereupon voltagecould be applied for final focusing. After completed final focusing thetop electrode is replaced by an attachment having a tight threadedstopper, and the stopper which till then has been situated in thetreaded hole 5 is replaced by a threaded outlet tube having a regulatingvalve. The stopper in the hole 6 now can be removed. The column isslightly tilted in order that the mouth of the hole 5 will be the lowestpoint of the column. After that the column can be emptied from itscontent, which is at that divided into fractions for pH-measurement andchemical and/or biological analysis.

The column can alternatively be made of quartz, whereby UV-absorptionanalysis can be accomplished directly in the column during theperforming of focusing, which is of great advantage. The fastening ofthe column wall 9 by screws then must be substituted by a device forclamp fastening, but besides, the same construction can be retained. Twodifferent sensitivity degrees in the optical analysis can be achieved byallowing the light to pass the path b or the path 0 in the column. Theshift between these degrees of sensitivity is simply accomplished byturning the column To utilize mercury as anode can be associated withcertain risks if it is considered that mercury ions could go intosolution and migrate through the column and transform the proteins inthe column by complex formation. It is true that the mercury I halidesare as sparingly soluble that they would hardly give rise to deleteriousconcentrations of mercury I ions, but at the anodical oxidation ofmercury also small amounts of mercury II salts, which are soluble areformed. However, these are so weakly dissociated that their cathodicalmigration will be very slow. Anyhow, there are doubtless cases in whichmercury anode is completely inconceivable. The combination column thenmust be used with the cathode in the bottom and a sufficient reserve ofalkali metal ions, or mercury as an electrode has to be completelyabandoned. In the latter case the column construction has to bemodified.

Principally this can be made by a symmetrical design in which the twocolumn walls having an internal area of ac units of surfaces are madedetachable as described in FIG. 1 for one of the walls. Thepalladiumsilver-anode then should be saturated with hydrogen and thepalladium-silver-cathode be free of hydrogen, and both the electrodesshould be removed after prefocusing. A remaining problem then is how torealize a non-gasing bottom electrode with an area of be units ofsurface during the final focusing. Presumably, the best way to achievethis is to prepare an extra column wall having the internal measure acunits of surface and in one end of this constructional element attach apal]adium-silver-electrode, which is slightly smaller than be units ofsurface. When the prefocusing is completed, the bottom electrode ispulled out with a temperary loosening of the screws 10, and then the topwall with its electrode is completely removed and substituted by theextra wall which in its one end makes the attachment for the bottomelectrode in final focusing position.

FIG. 2 shows a column device comprising a prefocusing column 21 and afinal focusing column 25in the position of the device during thetransport of prefocused material from one column to the other and duringthe final focusing. During the prefocusing the device is turned 90around an axis perpendicular to the plane of the paper so that the finalfocusing column 25 then will have a horizontal axis. The final focusingcolumn 25 in this example is represented by the above mentionedapparatus, marketed by LKB-Produkter AB. As described above, thisapparatus comprises in addition to the column noble metal electrodes, acentral gas outflow tube as well as a cooling jacket.

As these constructional details are known as such and are not part ofthe invention, only the outer contours of this column have been drawn inthe figure. The volumes of the two columns are equal.

The central part of prefocusing column is the focusing chamber 21itself, limited at the top and at the bottom by two electrolyticallyconducting but convection breaking membranes 24, e.g. glass filterdiscs. Laterally the focusing chamber is restricted by vertical walls,e.g. by a short elliptical or circular cylinder. In two diametricallyopposite points there are threaded holes 30, 31 in which alternativelythreaded stoppers or tubes can be inserted. During prefocusing there isin the hole 30 a stopper, in the hole 31 a tube which via a cock 32forms the connection to the final focusing column.

Outside the membrane 24 there is an anolyte chamber 22 or 23 and acatholyte chamber 23 or 22 which via tubes and circulation pumps 29 isconnected each to one electrode vessel 26,28 containing each anelectrode 27. Each tube-pipe comprises a coil, immersed into a coolingreservoir, which details, however, are not shown in the figure. Asuitable clamping device, which is neither shown, is holding in aleakage free manner anolyte chamber, focusing chamber, and catholytechamber together.

By operation of this column device first the membranes 24 are thoroughlymoistened in order to be im' permeable to air. Then into the hole 30 atube is inserted which is connected to a density gradient mixing device,and the cock 32 is opened. With the whole device in final focusingposition a vacuum is applied to the final focusing column in order thatthe prefocusing chamber 21 is filled with a solution of desired densityvariation. The cock 32 is closed, and the tube in the hole 30 issubstituted by a stopper. Whole the device is turned around an axisperpendicular to the plane of the paper, the electrode vessels arefilled with anolyte and catholyte, respectively, and the circulationpumps 29 are started. Voltage is applied to the electrodes forprefocusing.

After completed prefocusing the current is disconnected, and the deviceis carefully turned 90 around an axis perpendicular to the plane of thepaper. Anolyte and catholyte then are allowed to flow out through thegas outlet holes of the electrode attachments. It is also possible tocompletely remove the electrodes with their attachments. After thedevice has been raised the stopper in the hole 30 is substituted by atube, filled with and dipping into a fluid having greater a density thanthe solution in the bottom of the column. This tube should also beprovided with a cock or, if rubber or plastic tubes are used, a ligatureclamp. With the latter one closed, the cock 32 is opened. By applying asuitable vacuum to the final focusing column 25 and by governing thecock or ligature clamp of the tube inserted in 30, the column content inthe chamber 21 is replaced by the said heavier fluid, and the prefocusedmaterial then will ascend into the final focusing column, the coolingjacket of which during this operation being throughflown by coolingmedium. When the fluid transport is completed, voltage is applied to thefinal focusing column after the cock 32 has been closed. The prefocusingcolumn is removed, and after the final focusing the column content canbe fractioned by careful outflow through the cock 32.

FIG. 3 shows a column device distinguished from that one shown in FIG. 2only concerning the cooling system. In this device the anolyte andcatholyte chambers are made larger in order that there is room forcooling coils 33,34 through which cooling medium from a coolingreservoir is made to circulate. This device has the advantage comparedto that one shown in FIG. 2 that the circulation pumps are not under avoltage. The electrodes 35 can be attached in rather slender tubes 36,37which are centrally leading out into the anolyte and catholyte chambers.

The utilization of column devices according to FIG. 2 and FIG. 3 have incommon that the anolyte should consist of a solution of a strong acidand the catholyte of a solution of a strong base. Their concentrationsshould be between 0.1 and l equivalents per liter. Their conductivitiesthen will be several l0-powers greater than that one prevailing in thecentral parts of the column at focusing. This is why it is possible inFIG. 2 and 3 to have as narrow tubes for supplying of current to theanolyte and catholyte chambers. There is no risk of uneven currentdensity caused by the local falling out of the tubes into the anolyteand catholyte chambers.

FIG. 4 shows how a column device could be arranged in which theelectrodes are in direct contact with the prefocusing column and inwhich the fluid transport between the two columns is carried out fromthe top downwards. The figure is showing the device being in itsposition during fluid transport from one column to the other and duringfinal focusing. During the prefocusing the device is turned 90 around anaxis perpendicular to the plane of the paper, in order that the finalfocusing column 48 then will have a horizontal axis. Also in thisexample the final focusing column is represented by the above mentionedapparatus marketed by LKB-Produkter AB. As described above, thisapparatus comprises, besides the column itself, noble metal electrodes,a centrally situated gas outflow tube, and a cooling jacket. As theseconstructional details are known as such and are not making part of theinvention, only the outer countours of this column have been drawn inthe figure. The volumes of the two columns are equal.

The prefocusing column 52 is at its top and bottom limited by twonon-gasing electrodes 42 and 46, each joint by soldering to one externalwall of two cooling chambers 47 and 49, each provided with two pipesockets 41. Laterally the prefocusing column is restricted by verticalwalls 44, e.g. being a short elliptical or circular cylinder. In twodiametrically opposite points there are threaded holes 45,50 in whichalternatively threaded stoppers or tubes could be inserted. Duringprefocusing there is in the hole 45 a stopper, in the hole 50 a tubewhich via a cock 51 forms the connection to the final focusing column48. The prefocusing column is tightened by means of O-rings 43 combinedwith a clamping device acting on the outer walls of the two coolingchambers.

When operating this column device it is first placed in final focusingposition (as in the figure), the final focusing column is filled with afluid with greater a density than the bottom solution to be used in theprefocusing column, whereupon the bottom cock of the final focusingcolumn and the cock between the columns are closed. The prefocusingcolumn then is filled with an amfolyte solution containing the proteinsample, out of a density gradient mixer, whereupon the hole 45 is closedby means of a stopper. The column device then is turned slowly 90 aroundan axis perpendicular to the plane of the paper, the cooling water flowis started and electrical voltage is applied for prefocusing. Aftercompleted prefocusing the current is disconnected and the column deviceis turned backwards 90. The cocks in both ends of the final focusingcolumn are opened, and the stopper in the hole 45 is removed. By meansof one of the cocks the flow rate downwards is adjusted to a suitable,low value. After the fluid content of the prefocusing column has floweddown into the final focusing column, the bottom cock of the latter isclosed, and the prefocusing column is removed. Voltage is applied to thefinal focusing column, and after completed final focusing the current isdisconnected and the column content is divided into fractions flowingout through the bottom cock.

In a typical experiment with the device according to FIG. 1 theprefocusing required 1.5 hours and the final focusing 3 hours.

In an experiment with the device according to FIG. 4 the prefocusing wascarried out during 1 hour, whereupon the column content was transferredto the final focusing column during minutes. The final focusing was thencarried out during 5 hours. By using any of these apparatuses it ispossible to obtain the resolution power ApI 0.02, stated above.

With the device according to FIG. 4 it is most likely that thisresolution can be achieved in a prefocusing period of only 10 minutesand a final focusing period of 3 hours. Thus, the focusing period isdecreased from 24-48 hours to about 3.5-6.5 hours. Isoelectric focusingwith this high resolution in this short period of time means a decidedimprovement compared to hitherto known technique.

We claim:

1. Apparatus for the separation of components in a sample solution byisoelectric focusing in which said focusing is performed in at least twosuccessive focusing steps comprising, closed container means to bedisposed in a first position during said first focusing step and in asecond position during the second focusing step, the interior thereofdefining a focusing chamber, at least two pairs of oppositely disposedwalls, the mean area of one pair of said walls being less than the meanarea of the other pair of said walls, the mean distance between said onepair of walls being greater than the mean distance between said otherpair of walls, an electrode system for selectively generating a verticaldirect current electrical field across said sample between therespective inner surfaces of each pair of said two pairs of oppositelydisposed walls in either of said first or second positions of thecontainer means.

2. Apparatus according to claim 1 which includes means for externallycooling said focusing chamber.

3. The invention defined in claim 1, wherein said closed container meansis rotated bodily about a single axis for movement between said firstand second positions.

4. The invention defined in claim 1, wherein said electrode systemincludes a separate vessel contianing an electrode and an electrolytesolution therein, said separate vessel and said closed container meansbeing separated from each other by a convection breakingelectrolytically conducting membrane, said membrane defining at leastone of said two pairs of walls, said membrane separating the samplesolution from the electrolytic solution.

5. The invention defined in claim 4, wherein said separate vesselincludes means for cooling said electrolytic solution.

6. The invention defined in claim 5, wherein said means for cooling saidelectrolytic solution includes conduit means in said separate vessel forconducting a cooling medium in heat exchanging relationship through saidelectrolytic solution.

7. The invention defined in claim 1, wherein said electrode systemincludes at least one electrode element in direct contact with saidsample solution in one position of said container means.

8. The invention defined in claim 7, wherein said electrode elementcomprises palladium.

9. The invention defined in claim 7, wherein said electrode elementcomprises silver.

10. The invention defined in claim 7 wherein said electrode elementcomprises lead.

11. The invention defined in claim 7 wherein said electrode elementcomprises mercury.

12. The invention defined in claim 11 wherein said mercury electrodeelement is disposed at the bottom of said focusing chamber on arespective one of said two pairs of oppositely disposed walls in eitherof said first and second positions of the container means, said meansalso including means for cooling said mercury.

13. The invention defined in claim 7, wherein at least one wall of atleast one of said two pairs of walls includes a flat electrode extendingover the entire area of the inner surface of said one wall.

14. The invention defined in claim 13 wherein said closed containermeans includes a removable section, the inner surface of said sectiondefining said one wall, and means for detachably connecting said sectionin fluid tight engagement with the remainder of the container means.

15. The invention defined in claim 14 wherein said second position.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO.3,915,839 DATED October 28, 1975 INVENTOR(S) Svante Harry Rilbe and JarlSune Pettersson It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

On the cover page, please add the following:

-Poreign Application Priority Data May 16, 1972 SWEDEN ..6377/7Z- Signedand Scaled this second Day Of March 1976 [SEAL] Arrest:

RUTH- C. MA SON C. MARSHALL DANN Affeslmg ff Commissioner ofParents andTrademarks

1. APPARATUS FOR THE SEPARATION OF COMPONENTS IN A SAMPLE SOLUTION BYISOELECTRIC FOCUSING IN WHICH SAID FOCUSING IS PERFORMED IN AT LEAST TWOSUCCESSIVE FOCUSING STEPS COMPRISING, CLOSED CONTAINER MEANS TO BEDISPOSED IN A FIRST POSITION DURING SAID FORCUSING STEPS AND IN A SECONDPOSITION DURING THE SECOND FOCUSING STEP, THE INTERIOR THEREOF DEFININGA FOCUSING CHAMBER, AT LEAST TWO PAIRS OF OPPOSITELY DISPOSED WALLS THEMEANS AREA OF ONE PAIR OF SAID WALLS BEING LESS THAN THE MEAN AREA OFTHE OTHER PAIR OF SAID WALLS, THE MEAN DISTANCE BETWEEN SAID ONE PAIR OFWALLS BEING GREATER THAN THE MEAN DISTANCE BETWEEN SAID OTHER PAIR OFWALLS, AN ELECTRODE SYSTEM FOR SELECTIVELY GENERATING A VERTICALELECTRICAL FIELD ACROSS SAID SAMPLE BETWEEN THE RESPECTIVE INNER SURFACEOF EACH PAIR OF SAID TWO PAIRS OF OPPISITELY DISPOSED WALLS IN EITHER OFSAID FIRST SECOND POSITIONS OF THE CONTAINER MEANS.
 2. Apparatusaccording to claim 1 which includes means for externally cooling saidfocusing chamber.
 3. The invention defined in claim 1, wherein saidclosed container means is rotated bodily about a single axis formovement between said first and second positions.
 4. The inventiondefined in claim 1, wherein said electrode system includes a separatevessel contianing an electrode and an electrolyte solution therein, saidseparate vessel and said closed container means being separated fromeach other by a convection breaking electrolytically conductingmembrane, said membrane defining at least one of said two pairs ofwalls, said membrane separating the sample solution from theelectrolytic solution.
 5. The invention defined in claim 4, wherein saidseparate vessel includes means for cooling said electrolytic solution.6. The invention defined in claim 5, wherein said means for cooling saidelectrolytic solution includes conduit means in said separate vessel forconducting a cooling medium in heat exchanging relationship through saidelectrolytic solution.
 7. The invention defined in claim 1, wherein saidelectrode system includes at least one electrode element in directcontact with said sample solution in one position of said containermeans.
 8. The invention defined in claim 7, wherein said electrodeelement comprises palladium.
 9. The invention defined in claim 7,wherein said electrode element comprises silver.
 10. The inventiondefined in claim 7 wherein said electrode element comprises lead. 11.The invention defined in claim 7 wherein said electrode elementcomprises mercury.
 12. The invention defined in claim 11 wherein saidmercury electrode element is disposed at the bottom of said focusingchamber on a respective one of said two pairs of oppositely disposedwalls in either of said first and second positions of the containermeans, said means also including means for cooling said mercury.
 13. Theinvention defined in claim 7, wherein at least one wall of at least oneof said two pairs of walls includes a flat electrode extending over theentire area of the inner surface of said one wall.
 14. The inventiondefined in claim 13 wherein said closed container means includes aremovable section, the inner surface of said section defining said onewall, and means for detachably connecting said section in fluid tightengagement with the remainder of the container means.
 15. The inventiondefined in claim 14 wherein said closed container means also includes atleast one cooling chamber in heat exchanging relationship with saidsample solution.
 16. The invention defined in claim 1 wherein saidclosed container means comprises two focusing chambers in communicationwith each other, and valve means for confining said sample solution inone of said focusing chambers while in said one position and fortransferring the sample solution to the other of said focusing chamberswhen the container means is in said second position.